The present invention relates to a process for producing 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid for use in seasonings or the like from inosine or guanosine or a precursor thereof using adenosine triphosphate (ATP)-producing microorganisms containing a DNA encoding a protein that has the activity of forming 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid from inosine or guanosine.
Further, the present invention relates to a novel protein having the inosine-guanosine kinase activity, a gene encoding said protein, a recombinant DNA containing said gene, and a microorganism which is transformed with said recombinant DNA.
In order to produce 5xe2x80x2-inosinic acid by phosphorylating inosine using microorganisms, a method using p-nitrophenyl phosphate (Japanese Patent Publication No. 29,858/1964), a method using inorganic phosphoric acids (Japanese Patent Publication Nos. 1,186/1967 and 44,350/1974), a method using acetyl phosphate (Japanese Patent Application Laid-Open No. 82,098/1981), and a method using ATP (Japanese Patent Application Laid-Open No. 230,094/1988) have been developed so far. However, the accumulation of 5xe2x80x2-inosinic acid which is produced by these methods has not necessarily been satisfactory. As an improved method of phosphorylating inosine with ATP, a method which comprises obtaining a gene encoding inosine-guanosine kinase of Escherichia coli, preparing an E. coli strain having the increased inosine-guanosine kinase activity by recombinant DNA technique, and phosphorylating inosine or guanosine using this strain to produce 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid, has been also developed (WO 91/08286). However, this method requires that a microorganism for regenerating ATP to be consumed in the reaction is separately cultured and that its cells are added to the reaction solution. Accordingly, a method of obtaining 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid more efficiently has been in demand.
Moreover, it is only known that the inosine-guanosine kinase gene is presents in some microorganisms such as E. coli [J. Gen. Microbiol., 135, 1263-1273 (1989)].
The present inventors have developed a process for producing 5xe2x80x2-inosinic acid and/or 5xe2x80x2-guanylic acid more efficiently. Consequently, they have found that 5xe2x80x2-inosinic acid and/or 5xe2x80x2-guanylic acid can be produced easily in a high yield by introducing a gene encoding an inosine-guanosine kinase into a microorganism having sufficient ability to regenerate ATP to be consumed in the reaction. They have also found a novel inosine-guanosine kinase having an amino-acid sequence which is different from that of an inosine-guanosine kinase derived from E. coli. 
The present invention relates to a process for producing 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid for use in seasonings or the like from inosine or guanosine or a precursor thereof easily in a high yield. More specifically, the present invention is to provide a process in which a gene encoding a protein that has the activity of converting inosine and/or guanosine into 5xe2x80x2-inosinic acid and/or 5xe2x80x2-guanylic acid is introduced into a microorganism having a sufficient ability to regenerate ATP to be consumed in the reaction, whereby 5xe2x80x2-inosinic acid and/or 5xe2x80x2-guanylic acid is easily obtained efficiently in a high yield in the presence of only the microorganism having the gene introduced therein without separately culturing another microorganism for regenerating ATP to be consumed in the reaction and adding it to the reaction solution.
Further, the present invention is to provide a novel protein which can be obtained from microorganisms belonging to Exiguobacterium acetylicum and which has the activity of converting inosine and guanosine into 5xe2x80x2-inosinic acid and 5xe2x80x2-guanylic acid, respectively, a gene encoding said protein, a recombinant DNA containing said gene, a microorganism which is transformed with said recombinant DNA, and a process for producing 5xe2x80x2-inosinic acid and/or 5xe2x80x2-guanylic acid using this microorganism.
The protein of the present invention is novel in that the amino-acid sequence of the protein of the present invention is vastly different from that of a known protein having inosine-guanosine kinase activity. An inosine-guanosine kinase derived from E.coli has been already known. The present inventors have found that a protein having the inosine-guanosine kinase activity is also produced in microorganisms belonging to the genus Exiguobacterium which were not known before to have the inosine-guanosine kinase activity, and they have succeeded in isolating this protein and cloning the gene encoding the protein. This protein is much different from the known protein with respect to the amino-acid sequence.
It has been found for the first time by the present inventors that the protein having the amino-acid sequence quite different from that of the known protein having the inosine-guanosine activity has the same activity as the known protein.
That is, the present invention relates to the following:
(1) a process for producing 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid, which comprises contacting a transformant obtained by introducing a gene encoding a protein having inosine-guanosine kinase activity into a microorganism capable of reproducing ATP, with inosine or guanosine or a precursor thereof, an energy source and a phosphate donor, accumulating 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic arid in the reaction solution, and collecting the same therefrom,
(2) a process for producing 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid according to (1), wherein the microorganism capable of reproducing ATP belongs to a genus selected from the group consisting of Corynebacterium, Escherichia, Saccharomyces, Staphylococcus and Candida, 
(3) a process for producing 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid according to (1), wherein the microorganism capable of reproducing ATP belongs to Corynebacterium ammoniagenes, 
(4) a process for producing 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid according to any one of (1) to (3), wherein the gene encoding the protein having inosine-guanosine kinase activity is a gene derived from Exiguobacterium acetylicum or a gene capable of hybridizing said gene,
(5) a process for producing 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid according to any one of (1) to (3), wherein the gene encoding the protein having inosine-guanosine kinase activity is a gene derived from Escherichia coli or a gene capable of hybridizing said gene,
(6) a transformant obtained by introducing a gene encoding a protein having inosine-guanosine kinase activity into a microorganism capable of reproducing ATP,
(7) a transformant according (6), wherein the microorganism capable of reproducing ATP belongs to a genus selected from the group consisting of Corynebacterium, Escherichia, Saccharomyces, Staphylococcus and Candida, 
(8) a transformant according to (6), wherein the microorganism, capable of reproducing ATP belongs to Corynebacterium ammoniagenes, 
(9) a transformant according to any of claims 6 to 8, wherein the gene encoding a protein having inosine-guanosine kinase activity is a gene derived from Exiguobacterium acetylicum or a gene capable of hybridizing said gene,
(10) a transformant according to any of (6) to (8), wherein the gene encoding a protein having inosine-guanosine kinase activity is a gene derived from Escherichia coli or a gene capable of hybridizing said gene,
(11) a recombinant DNA being capable of replicating in Corynebacterium ammoniagenes and containing a gene encoding a protein having inosine-guanosine kinase activity,
(12) a recombinant DNA according to (11), wherein the gene encoding a protein having inosine-guanosine kinase activity is a gene derived from Exiguobacterium acetylicum or a gene capable of hybridizing said gene,
(13) a recombinant DNA according to (11), wherein the gene encoding a protein having inosine-guanosine kinase activity is a gene derived from Escherichia coli or a gene capable of hybridizing said gene,
(14) a protein obtainable from a microorganism belonging to Exiguobacterium acetylicum having inosine-guanosine kinase activity and the following characteristics:
1. Action
The enzyme transfers a phosphate group to a nucleoside in the presence of a phosphate donor and forms a nucleoside 5xe2x80x2-monophosphate.
2. Substrate Specificity
A phosphate group in the "Ugr"-position of a nucleoside triphosphate is transferred to the other nucleoside.
3. Optimum pH
pH 7.7-9.9.
4. pH Stability
pH 6.7-12.1.
5. Optimum Temperature
30-50xc2x0 C.
6. Metal Requirement
Magnesium ion, manganese ion, cobalt ion or iron ion
7. Influence of Metal Ions
The activity of the enzyme is strongly inhibited by copper ion and mercury ion, and is also inhibited by zinc ion and cadmium ion.
8. Km Values
Km value is 0.03 mM for guanosine, 1 mM for inosine, and 1.6 mM for ATP when guanosine is used as a substrate.
9. Molecular Weight
The enzyme has a molecular weight of approximately 36 kilodaltons as measured by SDS-polyacrylamide gel electrophoresis.
(15) a protein having inosine-guanosine kinase activity and having the amino acid sequence which is shown in SEQ ID NO:2 or in which a part of amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO:2,
(16) a gene encoding a protein according to (14) or (15), and
(17) a gene encoding a protein having inosine-guanosine activity, and having a nucleotide sequence shown in SEQ ID NO:1 or being capable of hybridizing a gene having said nucleotide sequence.
In the present specification, the activity of phosphorylating inosine and guanosine with ATP and forming 5xe2x80x2-inosinic acid and 5xe2x80x2-guanylic acid, respectively, is referred to as xe2x80x9cinosine-guanosine kinase activityxe2x80x9d. The protein having this activity is referred to as xe2x80x9can inosine-guanosine kinasexe2x80x9d. The microorganism having sufficient ability to regenerate ATP to be consumed in the reaction is referred to as xe2x80x9can ATP-producing strainxe2x80x9d.
The present invention will be described in more detail below.
The inosine-guanosine kinase referred to in the present invention is an enzyme that catalyzes the reaction of phosphorylating inosine and guanosine with ATP or the like to form 5xe2x80x2-inosinic acid and 5xe2x80x2-guanylic acid, respectively. The origin of this enzyme is not particularly limited but inosine-guanosine kinase derived from a microorganism is preferred. It includes not only a novel enzyme obtained from a microorganism belonging to Exiguobacterium acetylicum or the like but also a known inosine-guanosine kinase obtained from E. coli. 
The novel protein which can be obtained from a microorganism belonging to Exiguobacterium acetylicum or the like and which has the inosine-guanosine kinase activity can be obtained by culturing the microorganism, disrupting the obtained cells to prepare a crude enzyme extract, and purifying the enzyme from the crude enzyme extract. As an example of such microorganisms, Exiguobacterium acetylicum ATCC 953 can be mentioned.
Taxonomically, Exiguobacterium acetylicum had been called Brevibacterium acetylicum [Bergey""s Manual of Systematic Bacteriology, pp. 1301-1313 (1986)]. However, as a result of the genetic analysis, it is proposed that Brevibacterium acetylicum should be transferred to the genus Exiguobacterium as Exiguobacterium acetylicum [Int. J. Syst. Bacteriol., 44, 74-82 (1994)].
The inosine-guanosine kinase can be purified by any method that does not impair the inosine-guanosine kinase activity. The purification is generally performed through liquid column chromatography. Specifically, ion-exchange column chromatography using a potassium chloride concentration gradient, hydrophobic column chromatography using an ammonium sulfate concentration gradient, and adsorption column chromatography using a phosphate buffer concentration gradient may be used in combination.
In the present invention, the enzyme which can be obtained from the microorganism belonging to Exiguobacterium acetylicum and which has the inosine-guanosine kinase activity has the following properties.
1. Action
The enzyme transfers a phosphate group to a nucleoside in the presence of a phosphate donor and forms a nucleoside 5xe2x80x2-monophosphate.
The phosphate donor is a nucleoside triphosphate. Examples of the nucleoside triphosphate include ATP, 2xe2x80x2-deoxyadenosine triphosphate, guanosine triphosphate, 2xe2x80x2-deoxyguanosine triphosphate, and thymidine triphosphate.
Examples of the nucleoside to which the phosphate group is transferred include inosine, guanosine, and 21-deoxyguanosine.
The nucleoside 5xe2x80x2-monophosphate includes 5xe2x80x2-monophosphate esters of the above-mentioned nucleosides, and 5xe2x80x2-inosinate, 5xe2x80x2-guanylate, 2xe2x80x2-deoxy-5xe2x80x2-guanylate, etc. are given as the examples.
2. Substrate Specificity
A phosphate group in the y-position of the nucleoside triphosphate is transferred to the other nucleoside.
Examples of the nucleoside triphosphate include ATP, 2xe2x80x2-deoxyadenosine triphosphate, guanosine triphosphate, 2xe2x80x2-deoxyguanosine triphosphate, and thymidine triphosphate.
Examples of the other nucleoside to which the phosphate group is transferred include inosine, guanosine, and 2xe2x80x2-deoxyguanosine.
3. Optimum pH
The optimum pH is between 7.7 and 9.9.
4. pH Stability
The activity is stable in the pH range between 6.7 and 12.1.
5. Optimum Temperature
The optimum temperature is between 30 and 50xc2x0 C.
6. Temperature Stability
The enzyme is inactivated at 40xc2x0 C. or higher.
7. Metal Requirement
Metal ions are required to proceed with the reaction. The reaction proceeds in the presence of magnesium ion, manganese ion, cobalt ion or iron ion.
8. Influence of Metal Ions
The activity of the enzyme is strongly inhibited by copper ion and mercury ion, and is also inhibited by zinc ion and cadmium ion.
9. Km Value
Km value is 0.03 mM for guanosine, 1 mM for inosine, and 1.6 mM for ATP when guanosine is used as a substrate.
10. Molecular Weight
The enzyme has a molecular weight of approximately 36 kilodaltons as measured by SDS-polyacrylamide gel electrophoresis.
A DNA fragment containing the structural gene encoding the protein that has inosine-guanosine kinase activity can be obtained by a known method using a purified protein. Examples of the known method include a method in which an antibody against the above-mentioned protein is prepared and a chromosomal gene expression library is screened, and a method in which the amino-acid sequence of the protein purified is analyzed and the gene library is screened using a probe which is synthesized based on this amino acid sequence. As the amino acid sequence, an internal amino acid sequence of the protein determined from a polypeptide generated by an appropriate proteinase digestion of the protein, in addition to N-terminal amino acid sequence of the protein. Examples of the probe include oligonucleotides synthesized based on the N-terminal amino-acid sequence or the internal amino-acid sequence, those obtained by amplifying a region corresponding to the N-terminal amino-acid sequence or the internal amino-acid sequence through the polymerase chain reaction (PCR) using a oligonucleotide synthesized based on the sequence, and those obtained by amplifying the region corresponding to a portion from N-terminal to the internal amino acid using oligonucleotides synthesized based on the N-terminal amino-acid sequence and the internal amino-acid sequence as primers. Further, there is a method in which a chromosome is ligated with a double-stranded oligonucleotide which is called a cassette, and the desired fragment is obtained by PCR using a primer of an oligonucleotide formed according to the N-terminal amino-acid sequence and a primer formed according to the sequence of the cassette [Molecular and Cellular Probes, 6, 467 (1992)].
Specifically, a gene encoding a protein that has the inosine-guanine kinase activity of Exiguobacterium acetylicum can be obtained by amplifying a DNA fragment corresponding to the N-terminal region using PCR, synthesizing a primer based on the DNA fragment, and amplifying the fragment using PCR with the cassette.
The determined sequence of 28 amino acids in the N-terminal of the protein obtained from Exiguobacterium acetylicum ATCC 953 is represented by SEQ ID NO:3 in Sequence Listing. The 18th amino acid was not identified.
Judging form this N-terminal amino acid sequence, the protein of Exiguobacterium acetylicum is quite different from the known inosine-guanosine kinase derived from E. coli as described in WO 91/08286.
In order to obtain the aimed gene, the DNA encoding the N-terminal portion of the protein having the inosine-guanosine kinase activity is specifically amplified by PCR using the primer synthesized based on the above-mentioned N-terminal amino-acid sequence and using the chromosome of the microorganism belonging to Exiguobacterium acetylicum as a template, and is cloned. Ordinarily used is a primer in which the base composition is random, the G+C content is approximately 50%, no specific secondary structure is formed, the chains are not complementary to each other and the length is from 16 to 30 bases. The sequences of the primers are located at both terminals of the nucleotide sequence corresponding to the N-terminal region of the protein and are shown in SEQ ID No: 4 and 5 in Sequence Listing.
In SEQ ID No:4, the 6th nucleotide is a mixture of T and C, 9th nucleotide is a mixture of A and G, 12th nucleotide is a mixture of T, C and A, 15th nucleotide is a mixture of T, C, A and G. And in SEQ ID No:5, the 3rd and 12th nucleotides are a mixture of T and C, 6th nucleotide is a mixture of T, C, A and G, 9th and 15th nucleotides are a mixture of A and G.
Then, the chromosome of the microorganism belonging to Exiguobacterium acetylicum is cleaved with an appropriate restriction endonuclease. This cleaved substance is ligated with the cassette to form a template. A DNA fragment containing a structural gene portion or the upstream region of the protein having the inosine-guanosine kinase activity is specifically amplified by PCR using the above-mentioned template and the primer synthesized according to the nucleotide sequence corresponding to the N-terminal amino-acid sequence and the primer synthesized according to the cassette, and is cloned. The primer that satisfies the above-mentioned conditions, as shown in SEQ ID NO:6 and 7 in Sequence Listing, can be used.
A vector which is autonomously replicable in E. coli used as a host can be employed as a vector for cloning the gene. Examples of the vector include pUC19, pHSG298, pHSG398 and pBR322. Any strain which is suitable for the replication of the vector can be used as a recipient strain of the resulting recombinant DNA. Examples of the recipient strain include E. coli strains such as HB101, JM109 and DH5.
The nucleotide sequence of the inosine-guanosine kinase gene present in thee DNA fragment inserted into the vector and the amino-acid sequence of the protein encoded by this gene can be determined by analyzing the nucleotide sequence of this DNA fragment. The nucleotide sequence and the amino-acid sequence of the inosine-guanosine kinase of Exiguobacterium acetylicum ATCC 953 are represented by SEQ ID NO:1 and 2 in Sequence Listing, respectively.
The protein of the present invention comprises 303 amino acids, and the molecular weight is approximately 32.5 kilodaltons.
The protein of the present invention includes not only the protein represented by SEQ ID NO:2 in Sequence Listing but also proteins obtained from other strains belonging to Exiguobacterium acetylicum and other natural mutants that have the inosine-guanosine kinase activity.
Further, it is clear for a skilled person that proteins in which a part of the amino-acid sequence is substituted or deleted, proteins in which amino acids are added thereto and partially modified proteins may be used so far as these proteins have the inosine-guanosine kinase activity.
Instead of the gene derived from Exiguobacterium acetylicum, a gene which is capable of hybridizing the gene can be used so far as it encodes the inosine-guanosine kinase.
The gene which is capable of hybridizing the gene derived from Exiguobacterium acetylicum can be obtained from the following strains.
Exiguobacterium aurantiacum ATCC 35652
Kurthia gibsonii ATCC 43195
Kurthia zopfii JCM 6101.
The gene, as mentioned above can be obtained by a known method using the homology. Specifically, the following method can be employed.
First, the chromosomal DNA of any of the above-mentioned microorganisms is cleaved with an appropriate restriction endonuclease, and the cleaved fragments are subjected to agarose gel electrophoresis. The cleaved fragments are blotted on an appropriate transfer filter. The homologous fragments are detected by the Southern hybridization using the inosine-guanosine kinase gene derived from Exiguobacterium acetylicum as the probe to determine the length.
Among the fragments cleaved with the restriction endonuclease, the fragments having the aimed length is purified. The purification is generally conducted through sucrose density gradient centrifugation or recovery from an agarose gel with a glass powder. The thus-purified fragments are ligated with an appropriate vector, and an E. coli strain is transformed with the thus-obtained recombinant vector. The clone containing the aimed fragment having the inosine-guanosine kinase gene can be selected from among the resulting transformants using the colony hybridization method.
In the present invention, a gene encoding a known inosine-guanosine kinase can be used instead of the above-mentioned gene encoding the novel-inosine-guanosine kinase.
As the known inosine-guanosine kinase gene, the gene derived from E. coli can be used [J. Gen. Microbiol., 135, 1263-1273 (1989); J. Bacteriol. 177, 2236-2240 (1995)], and it can be obtained from, for example, E. coli ATCC 27325.
The known gene encoding the inosine-guanosine kinase can be obtained using a known method. The gene encoding the inosine-guanosine kinase which can be used in the present invention can be also obtained from a chromosomal DNA of E. coli ATCC 27325 using the following method.
First, primers are synthesized according to the sequence of the inosine-guanosine kinase gene derived from E. coli as represented by SEQ ID No:10 in Sequence Listing (WO 91/08286). Ordinarily used are primers in which the base composition is random, the G+C content is approximately 50%, no specific secondary structure is formed, the chains are not complementary to each other and the length is from 15 to 30 bases. The sequences of the primers are located at both terminals of the inosine-guanosine kinase structural gene as shown in SEQ ID Nos. 13 and 14.
Then, the inosine-guanosine kinase structural gene can be amplified from the chromosomal DNA of E. coli by PCR using these primers and cloned. A vector derived from E. coli, such as pUC19 and pBR322 is used. Any recipient strain which is suitable for the replication of the vector may be used for the resulting recombinant DNA. Examples of this recipient strain include E. coli strains such as HB101, JM109 and DH5. In this manner, the recombinant vector having the insertion of the DNA fragment containing the inosine-guanosine kinase gene of E. coli is obtained.
A gene which is homologous to the above-mentioned gene derived from E. coli and is capable of hybridizing the gene can be used, as in Exiguobacterium acetylicum, so far as it encodes the inosine-guanosine kinase.
The thus-obtained DNA fragment containing the gene encoding the protein that has the inosine-guanosine kinase activity is introduced into a host cell which can regenerate ATP after recombining it again with the other suitable vector or inserting the replication origin.
A microorganism which has the sufficient ability to regenerate ATP to be consumed in the reaction from an ATP precursor (ATP-producing ability) is used as the host cell.
In the present invention, the microorganism having the ATP-producing ability may be any microorganism having the ability to regenerate ATP to be consumed in the reaction of converting inosine and/or guanosine into 5xe2x80x2-inosinic acid and/or 5xe2x80x2-guanylic acid from the ATP precursor in the reaction system whereby the reaction can proceed. Examples of this microorganism include microorganisms belonging to the genus Corynebacterium, Escherichia, Staphylococcus, Saccharomyces or Candida. The microorganisms belonging to Corynebacterium ammoniagenes which have a high ability to produce ATP are especially preferable. Incidentally, Corynebacterium ammoniagenes was classified before as Brevibacterium ammoniagenes. 
Specific examples of the microorganisms having the ATP-producing ability which are used in the present invention are strains shown below and mutants derived therefrom.
Corynebacterium ammoniagenes (former name: Brevibacterium ammoniagenes) ATCC 6872
Corynebacterium ammoniagenes (former name: Brevibacterium ammoniagenes) ATCC 21295
Corynebacterium ammoniagenes (former name: Brevibacterium ammoniagenes) ATCC 21477
Corynebacterium glutamicum ATCC 13020
Corynebacterium glutamicum (former name: Brevibacterium flavum) ATCC 14067
Corynebacterium glutamicum (former name: Brevibacterium lactofermentum) ATCC 13869
Escherichia coli B (ATCC 11303)
Saccharomyces cerevisiae ATCC 20018
Staphylococcus aureus ATCC 4012
Candida zeylanoides ATCC 20356
Candida psychrophila (former name: Torulopsis psychrophila) ATCC 22163
Further, the microorganisms having the ATP-producing activity wherein the degrading activity of inosine and/or guanosine is weak or deficient are preferable. The following microorganisms are taken up from among the above-mentioned microorganisms.
Corynebacterium ammoniagenes ATCC 21295
Corynebacterium ammoniagenes ATCC 21477
As the ATP-producing microorganisms in the present invention, strains having the ability to produce inosine or guanosine from the precursor of inosine or guanosine in addition to the ATP-producing ability can be also used. In this case, 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid can be produced from the inosine or guanosine precursor instead of inosine or guanosine. Examples of this precursor include saccharides such as glucose, sucrose, molasses and starch hydrolysate, organic acids such as acetic acid, and alcohols such as glycerol and ethanol.
The ATP-producing strains having the ability to produce inosine or guanosine include:
Corynebacterium ammoniagenes ATCC 21478
Corynebacterium ammoniagenes ATCC 21479
Corynebacterium ammoniagenes ATCC 21480
The vector into which the gene encoding the inosine-guanosine kinase is integrated is not particularly limited so long as it can be replicated in the ATP-producing strains which are the recipient strains. For example, when bacteria belonging to the genus Corynebacterium are used as the ATP-producing strains, plasmids which can be autonomously replicated in these bacteria are mentioned. Specific examples thereof include pAM330 (Japanese Patent Application Laid-Open No. 67,699/1983), pHM1519 (Japanese Patent Application Laid-Open No. 77,895/1983), pAJ655, pAJ611, pAJ1844 (Japanese Patent Application Laid-Open No. 192,900/1983), pCG1 (Japanese Patent Application Laid-Open No. 134,500/1982), pCG2 (Japanese Patent Application Laid-Open No. 35,197/1983), pCG4, pCG11 (Japanese Patent Application Laid-Open No. 183,799/1982), pGA1 [Gene, 107, 69 (1991)], pHK4, and pHC4 (Japanese Patent Application Laid-Open No. 7,491/1993). When Escherichia coli is used as the ATP-producing strain, for example, Co1E1 plasmid, P15A plasmid, R-factor plasmid, F-factor plasmid and a phage plasmid can be used. Specific examples thereof include pBR322 [Gene, 2, 95 (1977)], pUC19 [Gene, 33, 103 (1985)], pACYC184 [J. Bacteriol, 134, 1141 (1978)], and pSC101 [Proc. Natl. Acad. Sci., U.S.A., 70, 3240 (1973)]. When Saccharomyces cerevisiae is used as the ATP-producing strain, YEp plasmid, YCp plasmid, YRp plasmid and YLp plasmid can be used. Specific examples thereof include YEp24, YRp7 and YCp5b. When Staphylococcus aureus is used as the ATP-producing strain, pRIT5 [EMBO J., 4, 1075 (1985)] can be used.
In order to express the gene encoding the inosine-guanosine kinase at high frequency, it is advisable that the promoter sequence and the SD sequence be located upstream of the gene encoding the inosine-guanosine kinase. A method of introducing these sequences is not particularly limited. The promoter sequence and the SD sequence can be introduced by a method in which the above-mentioned gene is inserted downstream of these sequences using the vector having these sequences, or a method in which these sequences are synthesized and inserted upstream of the above-mentioned gene; The promoter sequence and the SD sequence are not particularly limited. When the bacteria of the genus Corynebacterium are used as the ATP-producing strains, tac, lac and trp promoters derived from E. coli, trp promoter derived from bacteria of the genus Corynebacterium [Gene, 53, 191 (1987) ], fda promoter [Mol. Microbiol., 3, 1625 (1989)] . ppc promoter [Gene, 77, 237 (1989)], lysC promoter (Mol. Microbiol., 5, 1197 (1991) ], gdh promoter [Mol. Microbiol., 6, 317 (1992)], and csp1 and csp2 promoters (Japanese Patent Application Laid-Open 502,548/1994) can be mentioned. When Escherichia coli is used as the ATP-producing strain, tac, lac and trp promoters derived from E. coli, and PL promoter of xcex phage can be mentioned. When Saccharomyces cerevisiae is used as the ATP-producing strain, ADH1, ENO1, PGK1, GAP-DH, GAL1, GAL10, GAL7, PH05 and MFxcex11 promoters can be mentioned. When Staphylococcus aureus is used as the ATP-producing strain, spa promoter [J. Bacteriol., 159, 713 (1984)] can be mentioned.
A method of introducing into the ATP-producing microorganism the recombinant DNA containing the gene encoding the protein that has the activity of converting inosine and/or guanosine into 5xe2x80x2-inosinic acid and/or 5xe2x80x2-guanylic acid is not particularly limited. This introduction can be performed by a usual method. For example, when the bacteria of the genus Corynebacterium are used as the ATP-producing strain, the protoplast method (Japanese Patent Application Laid-Open No. 183,799/1982) and electroporation (Japanese Patent Application Laid-Open No. 207,791/1990) are especially effective. When Escherichia coli is used as the ATP-producing strain, the calcium chloride method [J. Mol. Biol., 53, 159 (1970)], the method of Hanahan [J. Mol. Biol., 166, 557 (1983)], the SEM method (Gene, 96, 23 (1990)], the method of Chung et al. [Proc. Natl. Acad. Sci., U.S.A., 86, 2172 (1989)], and electroporation [Nucleic Acids Res., 16, 6127 (1988)] can be used. There is a method in which a DNA is introduced by preparing competent cells from cells at the stage of proliferation as reported with respect to Bacillus subtilis [Gene, 1, 153 (1977)). Alternatively, a method in which cells of a DNA recipient strain are formed into protoplasts or spheroplasts that easily incorporate a recombinant DNA and the recombinant DNA is introduced into this DNA recipient strain, as reported with respect to Bacillus subtilis, actinomycetes, and yeasts [Molec. Gen. Genet., 1, 111 (1979), Nature, 274, 398 (1978), and Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)] can be also used. When Saccharomyces cerevisiae is used as the ATP-producing strain, the recombinant DNA can be introduced by the spheroplast method [Proc. Natl. Acad. Sci., U.S.A., 75, 1929 (1978)], the lithium acetate method [J. Bacteriol., 153, 163 (1983)], or electroporation [xe2x80x9cMethods in Enzymologyxe2x80x9d, 194, 182 (1991)]. When Staphylococcus aureus is used as the ATP-producing strain, the introduction of the recombinant DNA can be conducted by the protoplast method [Plasmid, 5, 292 (1981)] In the protoplast method, the high frequency can be obtained by the above-mentioned method which is used in Bacillus subtilis. However, as disclosed in Japanese Patent Application Laid-Open No. 183,799/1982, a method in which a DNA is incorporated into a state where protoplasts of cells of bacteria belonging to the genus Corynebacterium are brought into contact with either polyethylene glycol or polyvinyl alcohol and divalent metal ions can be also utilized. The uptake of the DNA can be enhanced by the addition of carboxymethyl cellulose, dextran, Ficol or Pluronic (made by Serva Co.) or the like instead of polyethylene glycol or polyvinyl alcohol.
The recombinant DNA can be introduced into the recipient strain by the electroporation method (refer to Japanese Patent Application Laid-Open No. 207,791/1990). The transformation method used in Examples of the present invention is electroporation.
Further, the inosine-guanosine kinase gene can be integrated into the chromosomal DNA of the ATP-producing microorganisms. The method of integrating this gene into the chromosomal DNA is not particularly limited. For example, a temperature-sensitive replication origin derived from bacteria of the genus Corynebacterium, an inosine-guanosine kinase gene and a marker which gives resistance to antibiotics such as chloramphenicol are inserted into a plasmid vector to form a recombinant DNA. The bacterium of the genus Corynebacterium is transformed with this recombinant DNA. The transformant is cultured in a medium containing antibiotics at a temperature at which the temperature-sensitive replication origin does not function, to form a transformant strain in which the recombinant DNA has been integrated into the chromosomal DNA [J. Bacteriol., 162, 1196 (1985), and Japanese Patent Application Laid-Open No. 7,491/1993]. Or a method using a movable genetic element derived from bacteria of the genus Corynebacterium can be also used [xe2x80x9cMobile Genetic Elementsxe2x80x9d, Academic Press, New York (1983), and WO 93/18151].
The inosine-guanosine kinase activity can be expressed at high level by culturing the thus-obtained transformant of the present invention into which the recombinant DNA containing the gene encoding the protein which has the inosine-guanosine kinase activity has been introduced, in an ordinary culture medium containing a carbon source, a nitrogen source, inorganic salts and optionally trace organic nutrients.
Examples of the carbon source include saccharides such as glucose, sucrose, molasses and starch hydrolysate; organic acids such as acetic acid and citric acid; and alcohols such as ethanol. Examples of the nitrogen source include urea, ammonium salts; aqueous ammonia and ammonia gas. Examples of the inorganic salts include phosphates, and potassium, magnesium, iron and manganese salts. Examples of the trace organic nutrients include amino acids, vitamins, fatty acids and nucleic acids as well as peptone, yeast extract and soybean protein hydrolysate containing any of these.
The cultivation is aerobically carried out at a temperature of from 25 to 37xc2x0 C. for 10 to 40 hours while adjusting pH between 5 and 9.
After the completion of the cultivation, the activity of the inosine-guanosine kinase accumulated in the culture is measured to confirm the titer. The activity can be measured by the method described in Molec. Gen. Genet. 143, 85-91 (1975) using a substance obtained by disrupting the cells recovered from the culture through centrifugation or the like using sonication or French-press treatment, centrifuging the disrupted cells to remove the cell residues, and removing low-molecular substances through gel filtration.
The culture of the microorganism containing the gene encoding the inosine-guanosine kinase and having the ability to biologically synthesize ATP from the ATP precursor, the cells separated from this culture, or the treated product of the cells is contacted with inosine or guanosine or the precursor thereof in the presence of the energy donor and the phosphate group donor, thereby forming 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid in the reaction solution. The cells can be separated from the culture through centrifugation or the like. The treated product of the cells includes acetone-treated cells, immobilized cells, disrupted cells, etc.
Materials which are preferably used in the present invention are mentioned below.
Examples of the precursor of inosine or guanosine include saccharides such as glucose, sucrose, molasses, starch hydrolysate, etc.; organic acids such as acetic acid. etc.; and alcohols such as glycerol, ethanol, etc.
Examples of the energy donor include saccharides such as glucose, sucrose, starch hydrolysate, molasses, etc.; organic acids such as acetic acid, citric acid, etc.; and alcohols such as ethanol, etc.
Examples of the phosphate group donor include inorganic phosphoric acids such as orthophosphoric acid, pyrophosphoric acid, polyphosphoric acid, tripolyphosphoric acid, polymethaphosphoric acid, hexamethaphosphoric acid, etc.; salts of these inorganic acids; and organic phosphoric acids such as phenyl phosphate, acetyl phosphate, carbamyl phosphate, etc.
The efficiency of the reaction can be improved by adding an ATP precursor, a surfactant, a metal ion, etc., to the reaction solution.
Examples of the ATP precursor include adenosine diphosphate, adenylic acid, adenosine, adenine, adenine mineral acid salt, a ribonucleic acid hydrolysate, etc.
The surfactant may be a cationic, anionic or amphoteric surfactant so far as it enhances the phosphorylation of inosine or guanosine. Examples of the metal ion include magnesium ion, manganese ion, etc.
In the ordinary phosphorylation reaction using a combination of an inosine-guanosine kinase and an ATP-producing strain, an organic solvent is generally added to the reaction system (Japanese Patent Application Laid-Open No. 230,094/1988 and WO 91/08286). Meanwhile, in the present invention, the reaction proceeds efficiently even when the organic solvent is omitted in the reaction system.
The reaction is aerobically performed at a temperature of from 25 to 37xc2x0 C. for 10 to 30 hours while adjusting pH between 6 and 8.
After the completion of the reaction, 5xe2x80x2-inosinic acid or 5xe2x80x2-guanylic acid accumulated in the reaction solution can be collected by ion-exchange resin treatment, crystallization, or the like.